Project Summary While mucus barriers provide natural protection from pathogens and allow for passage of nutrients, loss of homeostasis in diseases such as cystic fibrosis (CF) results in abnormal mucus secretions with dysfunctional clearance mechanisms. As a result, most morbidity and mortality in CF patients from advanced lung disease is in part due to the altered mucus microenvironment. In CF, the local mucus environment not only promotes the development of chronic bacterial infections, but it is hyperconcentrated and viscous, preventing effective penetration of therapeutics through the barrier to correct the affected epithelia or infections. To improve delivery and efficacy of therapeutics, it is critical to enhance therapeutic penetration through the mucus barrier. Current strategies have focused on hydrophilic, net-neutral charge polymers to improve transport and minimize interactions with mucus. However, current technology may be immunogenic after repeated dosing and may demonstrate suboptimal cellular uptake. Also, it is unclear if current technology achieves maximum transport, and studies have been limited to a small number of testable formulations with uniform surface chemistries, which may not be optimal interfaces for mucus penetration. Using bacterial viruses, i.e. bacteriophage, we have identified phage-presenting peptides from a large combination of random peptides (107-109) that are mucus-inert and facilitate transport through the mucus barrier. From this finding, the objective of this proposal is to develop chitosan nanoparticles that mimic mucus- penetrating bacteriophage and deliver CRISPR/Cas9 targeting defective CFTR mutations to treat cell culture and animal models of cystic fibrosis. We hypothesize that our chitosan nanoparticles will achieve effective complexation of CRISPR/Cas9 nucleic acids, and functionalization of these nanoparticles with mucus-inert peptides will successfully overcome the mucus barrier for effective gene delivery into the diseased epithelia. We further propose that the composition and distribution of penetrating peptides will change transport behavior in mucus. To test these hypotheses, the specific aims of this work will focus on the following: (1) develop chitosan CRISPR/Cas9 complexes coated with mucus penetrating peptides; (2) validate rapid transport of these nanoparticles by particle tracking microscopy and perform mutagenesis studies to dissect the amino acids responsible for facilitating mucus penetration; and (3) confirm delivery and gene correction of defective CF bronchial epithelium cell lines and animal model of CF. The proposed work is innovative because by mimicking mucus-penetrating bacteriophage, we will have developed a new class of synthetic chitosan nanoparticles capable of targeted genomic editing for therapy. The significance of the proposed work is bacterial viruses provided the inspiration for design of new gene targeting delivery systems, and from this work, we can begin to understand physicochemical properties impacts transport, and thus, will provide comprehensive design principles to develop more effective gene and drug delivery through mucus barriers.